Two low-k materials, dense and porous low-k films, were used. Experimental results indicated that the porous low-k films had shorter breakdown times, lower Weibull slope parameters and electric field acceleration factors, and weaker thickness-dependence breakdowns compared to the dense low-k films. Additionally, a larger derivation in dielectric breakdown projection model and a single Weilbull plot of the breakdown time distributions from various areas merging was observed. This study also pointed out that the porous low-k film in the irregular-shaped metal gate MIS capacitor had a larger dielectric breakdown time than that in the square- and circle-shaped samples, which violates the trend of the sustained electric field. As a result, another breakdown mechanism exists in the irregular-shaped sample, which is required to explore in the future work.In this study, the reliability characteristics of metal-insulator-semiconductor (MIS) capacitor structures with low-dielectric-constant (low- Currently, carbon-doped silicon (C-SiO2) film with the incorporation less polarizable Si–H or Si–R termination (where R is an organic moiety such as CH3), which is also called organosilicateglass (OSG) film, is widely used in the semiconductor industry. To further reduce the dielectric constant, porous low-k materials are produced by introducing nano-pores into the existing OSG low-k film. As the feature size of integrated circuits (ICs) is continuously decreased below 0.25 μm, the interconnect resistance/capacitance (RC) delay limits the performance of ICs ,2. To ovectively ,4. The lk films are integrated in the back-end-of-line Cu interconnects, more challenges arise due to poor mechanical and electrical properties for low-k films compared to the traditional SiO2 film. On the other hand, with the advance of technology node of ICs, the dimensions of BEOL interconnects are continuously shrinking. However, the supply voltage does not reduce by the same proportion. This leads to a higher electric field being subjected to the low-k insulator [k material reliability to become a bottleneck for new technology node qualification. To overcome this issue, therefore, understanding the reliability characteristics of low-k materials is essential and necessary as they are used as IMDs in ICs.As these low-nsulator ,6. Thesek materials is an important topic. This work investigated the reliability characteristics of low-k materials using metal-insulator-semiconductor (MIS) capacitor structures. The effects of metal gate area and geometry, thickness of a low-k film, and Cu drift were examined. Two kinds of low-k materials without and with porosity  were used. Therefore, the reliability of low-F is the cumulative failure probability and is a function of the random variable tbd for time-to-breakdown, β is the Weibull slope parameter which governs the breakdown distribution, and T63.2% is the characteristic dielectric breakdown time which represents the time for 63.2% sample failure. Plotting the Weibull function (W) can obtain the Weibull plot, given byBreakdown of a dielectric film breakdown is a statistical phenomenon and is usually described by the Weibull distribution (1)F. k films, respectively. Both dielectric films exhibited their breakdown times decreasing with increasing electric field, indicating that the electric field is an important factor in controlling breakdown of a dielectric film. Comparing the breakdown times of dense and porous low-k films indicates that the dense low-k film had a longer breakdown time than the porous film.MIS capacitors with dense and porous low-T63.2% ) for dense and porous low-k films films obtained from T63.2% decreased with increasing the stressing electric field. Moreover, the porous low-k films had the smaller β and T63.2% compared to the dense low-k film in a fixed electric field due to the existing pores in the films. According to electric field analyses within the porous dielectric film, the pores possess a higher field than the other sites [er sites , triggerα is a constant that depends on the properties of a dielectric film, γ is a field acceleration factor, E is the applied electric field, and m = 1 for E model and m = 1/2 for E1/2 model. Based on the E or E1/2 models, the field acceleration factors and the predicted lifetimes at 1 MV/cm and 2 MV/cm for the dense and porous low-k films are listed in 1/2 models, the extracted field acceleration factor was larger in the dense low-k film than in the porous low-k film. Moreover, both low-k films had a larger field acceleration factor when the E1/2 model was applied. Therefore, the predicted lifetimes in the low field were optimistic for the E1/2 model compared to the E model, which is consistent with other reports [E2) between the experimental data and fitting model are also listed in n is the total number of stress conditions, T63.2% are TTF at 63.2% failure for experimental data and fitting model results. The smaller E2 value represents the more accurate value for the fitting model. As listed in k films, indicating that both the E and E1/2 models are suitable for low-k film (SiCOH) TDDB reliability. Additionally, porous low-k films had a slightly larger error value, implying that the pores within the low-k film would alter TDDB behavior. In order to further verify the accuracy of TDDB model for low-k dielectric films, TDDB tests in lower electric fields (approaching operation electric field) will be performed in a future study.To precisely predict the lifetime of a dielectric film in a low operation electric field, an accurate TDDB model to describe dielectric reliability is of importance. A widely accepted TDDB model is of the general form ,12:(3)ln reports ,14. In ok film TDDB reliability because the dimensions of the interconnects can vary provided they comply with the design rule. In this part, the square-shaped metal gate MIS capacitors with areas of 0.1–2.5 × 10−3 cm−2 were used. Before TDDB tests, C-V measurements were made to calibrate the metal gate area by using the following Equation (5):C is the capacitance measured at 1 MHz, 0ε is absolute capacitivity in vacuum (8.85 × 10−12 F/m), k is the dielectric constant of a dielectric film, A and d represent the metal gate area and film’s thickness, respectively. C-V measurement results indicated that the capacitance decreased with increasing area of the designed metal gate. Assuming that the dielectric constant and thickness of a dielectric film remains constant without metal gate area independence, the actual area is therefore determined according to Equation (5). Here, the adopted dielectric constants of dense and porous low-k films were 3.02 ± 0.05 and 2.56 ± 0.08, respectively, which were determined from Hg probe measurement. Based on Equation (5) and the measured capacitances and dielectric constant, the areas of metal gate can be calibrated. The areas of metal gate in the fabricated MIS capacitors were higher than those of designed metal gate by 5~10% owing to shadow effect.Area effect is an essential topic for low-k films, respectively. For dense and porous low-k films, the stressing fields were fixed at 8.8 MV/cm and 7.1 MV/cm, respectively. As shown, TDDB TTFs decreased and the distributions became board with increasing metal gate area for both low-k films.The fabricated MIS structures with various metal gate areas were stressed at a fixed electric field in TDDB tests. A1 and A2, a Weilbull scale and two distributions, F1 and F2, are vertically shifted based on the following Equation (6):The breakdown events satisfy Poisson random statistics independently of the use of cumulative density distribution because the breakdown sites are randomly distributed over the capacitor area . As the −4 cm2 based on Equation (6), as plotted in k films, respectively. For dense low-k films, their TDDB TTFs in various areas can be merged to a single Weilbull distribution, suggesting that the breakdown is intrinsic and the failure site is randomly distributed across the dielectric film. On the other hand, for porous low-k films, the transformed distribution deviated from a single Weilbull distribution, especially for a larger area. The obtained TDDB features in the porous low-k film are strongly associated with the pores. For the porous low-k film in the capacitor with a larger area, the pores are likely to be highly connected, thereby accelerating breakdown. From the merge Weilbull distributions, the β values were determined to be 1.57 and 1.20 for dense and porous low-k films, respectively. The determined β value is very useful to forecast the failure times of a dielectric film in the capacitors with various areas according to the following relation:The measured failure times were normalized corresponding to an area of 1.0 × 10T63.2% values of MIS capacitors with various areas for dense low-k and porous low-k films. The measured T63.2% values at three metal gate areas are also presented for comparison. It is evident to note that the predicted T63.2% values are fitted well with the experimental data (within 10% error), demonstrating that Poisson area scaling is valid for the studied dielectric films.k film. The actual metal area was also calibrated by C-V measurements. The stressing field in the TDDB test was fixed to be 7.1 MV/cm. To compare TDDB performance for various metal gate geometries, the measured TTFs were correlated based on the metal gate area of 1.60 × 10−3 cm2 according to Equation (7). T63.2% for MIS capacitors with various metal gate geometries. The order of TTF was: Irregular > Circle > Square.TDDB tests were also carried out on the circle- and irregular-shaped MIS capacitors with the porous low-k films in the square-shaped MIS capacitors sustain a higher electric field, resulting in shorter TTFs. By ANSYS simulation, the sustained electric field within the porous low-k film in the MIS capacitors with various metal gate geometries can be obtained, as shown in As a constant voltage is applied on the square-shaped MIS capacitors during a TDDB test, it is likely to produce a higher electric field at the edge of metal gate due to the effect of point discharge. As a result, the porous low-k film in the irregular-shaped MIS capacitors had the highest maximum sustained electric field, possibly due to more edges, but had the longer TTFs than that in the circle- and square-shaped MIS capacitors. As shown in k dielectric’s reliability. In order to explore this mechanism, further experiments will be conducted in the future.On the other hand, the porous low-k films with thicknesses from 100 to 550 nm were used to test. The geometry of the tested MIS capacitors was square-shaped with an area of 9.0 × 10−4 cm2. The breakdown field was determined from I-V measurements. EB) versus the physical thickness for dense and porous low-k films. The breakdown field increased with decreasing the thickness for both low-k films. Under electric stress, charge trapping and de-trapping simultaneously occur in a dielectric film. As the trapped charges reach the critical value to form a conductive path within a dielectric film, breakdown occurs. As a result, as the thickness of a dielectric film decreases, de-trapping is becoming active, leading to an increase of breakdown field. As the thickness of a dielectric film is further scaling-down, the de-trapping rate would exceed the trapping rate, making a dielectric breakdown not occur [dc) at which the de-trapping rate of charges equals the trapping rate. A dielectric film whose thickness is less than this critical thickness does not exhibit dielectric breakdown. Since the dielectric breakdown strength is inversely proportional to the physical thickness, the thickness-dependent dielectric strength can be expressed as Equation (8):A is the constant, d is dielectric physical thickness, and n represents the power-law constant. The fitted n values from the slope of the line in k films, respectively. These obtained values were in the reported range of 0.2~1.0 [k films had a higher n value than the porous low-k films, indicating that the breakdown strength of the dense low-k films is strongly affected by their thicknesses. For the porous low-k film, its breakdown strength is degraded by the existing pores. Hence, the impact of the thickness on the breakdown strength is not as strong as that of the dense low-k film. The obtained critical thicknesses of the dense and porous low-k films, which were calculated using the least-squares method, were 12.90 nm and 7.86 nm, respectively. For the porous low-k films, the existing pores act as conducting sites, thereby reducing the critical number of electronic charges that are required to form a conducting path between the cathode and the anode and leading to a lower critical thickness.In this part, the dense and porous low-ot occur . Accordi 0.2~1.0 . The denk films with thicknesses of 100~560 nm were measured. The E-model was used to predict the failure time in a low operating electric field of 1.0 MV/cm. In order to obtain γ values, three fields were stressed for each condition. The TDDB results are shown in The breakdown times of the dense and porous low-k dielectric films had larger γ values as than porous low-k samples. Additionally, the obtained γ values increased as the physical thickness decreased. A larger γ value can result in a longer dielectric failure time in a lower electric field. Based on the TDDB results, the dielectric breakdown times of the dense and porous low-k films in an electric field of 1.0 MV/cm, predicted using Equation (3), as a function of thickness are plotted in k films increased with decreasing the film thickness. Additionally, the dense low-k films had longer dielectric breakdown times in an electric field of 1.0 MV/cm than did the porous low-k dielectric films. This improvement ratio of the dielectric breakdown time between dense and porous low-k dielectric films increased as the dielectric film became thinner. Since a thinner low-k film exhibited a pronounced de-trapping effect, the thickness-dependent dielectric breakdown time in an electric field of 1.0 MV/cm satisfies the dielectric strength equation, as given by Equation (9):f is the constant, and m represents the power-law constant. The cd values of the dense and porous low-k dielectric films, obtained from Equation (7), were 12.90 nm and 7.86 nm, respectively. Based on the predicted dielectric breakdown times and Equation (9), the extracted m values of the dense and porous low-k dielectric films were 2.868 and 1.067, respectively.The dense low-k film were deposited on 300 mm p-type Si wafers using applied material plasma-enhanced chemical-vapor-deposition reactor with a radio frequency (rf) of 13.56 MHz at a temperature of 300 °C. Both low-k films, which use diethoxymethylsilane (DEMS) and oxygen (O2) as the deposition matrix precursors, were SiCOH materials. To produce porous low-k films, alpha-terpinene (ATRP) was also introduced into the reactor as a porogen precursor. After deposition, UV thermal-assisted curing with 200~450 nm wavelength was performed to remove the organic porogen to form the pores in the film. Details about the deposition conditions of dense and porous low-k films can be found elsewhere [k films are listed in Both dense and porous low-lsewhere ,16. The k films was analyzed on an optical-probe system with an ellipsometer (Film TekTM 3000SE). The pore size and porosity were determined from the isotherm of ethanol adsorption and desorption using ellipsometric porosimetry . The dielectric constants of films were calculated from capacitances of the MIS structure at the voltage of 0.1 V and the frequency of 1 MHz using a semiconductor parameter analyzer (HP4280A).The thickness and refractive index of both low-I-V), capacitance voltage (C-V) characteristics, and dielectric-dependence dielectric breakdown (TDDB) failure time. The measurement tools used were a semiconductor parameter analyzer (HP4280A) and an electrometer . All measurements were performed at room temperature (25 °C).After the deposition of dielectric films, a metal electrode (Al) with a thickness of ~10 nm was deposited on the top surface of the films through shadow mask using a thermal evaporation method. The patterns used in the shadow mask are listed in k films have been investigated and compared in this study. The effects of metal gate area and geometry and low-k film thickness are studied. Compared to the dense low-k film, the porous low-k film has a shorter TDDB lifetime, a lower β value and electric field acceleration factor, and a weaker thickness-dependence breakdown. Additionally, it has a larger deviation in the breakdown projection model (E and E1/2 models) and single Weilbull plots of the breakdown time distributions from various areas merge. Finally, the dielectric breakdown time is also affected by the geometry of the metal gate. The porous low-k film in the irregular-shaped metal gate MIS capacitor had a larger dielectric breakdown time than that in the square- and circle-shaped samples, which violates the trend of the sustained electric field. A clear breakdown mechanism for the irregular-shaped sample has not been made clear. Hence, further experiments will be carried out in the future.The reliability characteristics of dense and porous low-


C. elegans as a model organism for detecting the toxic potency of whole influent wastewater samples. Gene expression in the nematode was used as bioanalytical tool to reveal the presence, type and potency of molecular pathways induced by 24-h exposure to wastewater from a hospital (H), nursing home (N), community (C), and influent (I) and treated effluent (E) from a local wastewater treatment plant. Exposure to influent water significantly altered expression of 464 genes, while only two genes were differentially expressed in nematodes treated with effluent. This indicates a significant decrease in bioactive pollutant-load after wastewater treatment. Surface water receiving the effluent did not induce any genes in exposed nematodes. A subset of 209 genes was differentially expressed in all untreated wastewaters, including cytochromes P450 and C-type lectins related to the nematode’s xenobiotic metabolism and immune response, respectively. Different subsets of genes responded to particular waste streams making them candidates to fingerprint-specific wastewater sources. This study shows that gene expression profiling in C. elegans can be used for mechanism-based identification of hydrophilic bioactive compounds and fingerprinting of specific wastewaters. More comprehensive than with chemical analysis, it can demonstrate the effective overall removal of bioactive compounds through wastewater treatment. This bioanalytical tool can also be applied in the process of identification of the bioactive compounds via a process of toxicity identification evaluation.With chemical analysis, it is impossible to qualify and quantify the toxic potency of especially hydrophilic bioactive contaminants. In this study, we applied the nematode The online version contains supplementary material available at 10.1007/s00244-022-00959-y. A multitude of chemical substances used for anthropogenic activities often end up in municipal wastewater  was proposed for reliable and reproducible data. Karengera et al. water samples were centrifuged and filtered to remove suspended solids. This implies that mainly water-soluble pollutants were present in samples after filtration with only a limited contribution from moderately hydrophobic compounds.In the present study, we aim to evaluate the applicability of the Wastewater samples were obtained from the sampling campaign as described by Verburg et al. . Briefly® Hydrophilic PTFE (0.45 µm pore size). For all filtrates, pH values in a range of 8.5–9.8 were measured prior to the use for the nematodes exposure. C. elegans has been shown previously to be tolerant to such test conditions . Next, the supernatants were further filtrated using Syringe filters MillexC. elegans wild-type Bristol N2 strain were cultured and exposed in three biological replicates for 24 h as described by Karengera et al.  and flash-frozen in liquid nitrogen for 1 min before storing them at − 80 °C until the extraction of RNA.Synchronized L4 stage larvae of a et al. . Prior t® Reagent with the PureLink® RNA Mini Kit was used to extract total RNA as described by Karengera et al. . The RNA was subsequently isolated from the crude extracts following the manufacturer’s protocol (Thermo Fisher MAN0000406) including column-based RNA isolation through binding, washing, and elution steps. A NanoDrop spectrophotometer was used to measure RNA quantity and quality (Table S1), with an A260/A280 ratio of 1.8 to 2.0 as requirement for further use.TRIzola et al. . BrieflyMicroarray analysis was conducted as described before by Karengera et al.  includinC. elegans tubulin gamma chain (tbg-1) and 14-3-3-like protein (par-5) as housekeeping genes.Gene expression of fifteen target genes selected from microarray data was tested by using RT-qPCR. The cDNA was synthesized from RNA templates via reverse transcription (RT) by using SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme as described by Karengera et al. . Two biop value < 0.0001 was considered as statistically significant. Custom written scripts for the microarray analysis are provided at https://git.wur.nl/published_papers/karengera_2021_wastewater_fingerprinting. To analyze the variation in gene expression, principal component analysis (PCA) was applied on the log2 ratio with the mean expression values using the prcomp function in “R”  in RStudio (version 1.1.463).Microarray data were statistically analyzed as described by Karengera et al. . Brieflyspl-2) genes were found to be the most upregulated transcripts for all wastewater samples. Expression of sphingosine phosphate lyase encoded by spl-2 is involved in defense responses to gram-positive bacterium. The function of protein encoded by C23G10.11 is not yet known.The exposed and unexposed nematodes did not show lethality for all tested water samples, as confirmed by visual observation through a stereomicroscope. Whole-transcriptome analysis using microarrays revealed a clear difference between the gene expression patterns induced by wastewater samples before and after wastewater treatment Fig. . Based o10(p) > 4.0; false discovery rate, FDR < 0.01). In contrast, differential expression in samples H and I was much lower with 464 and 406 genes, respectively. Only two genes (ncx-4 and F22B8.7) were differentially expressed in the nematodes treated with sample E and were both upregulated (1.1-fold for ncx-4 and 1.5-fold for F22B8.7). Of these two genes, differential upregulation of F22B8.7 (1.4-fold) was also found in the sample I. Of the genes whose transcription levels  were changed more than fivefold  were upregulated, while the other 26 genes were downregulated. Many upregulated genes were related to the nematode metabolic processes, especially those involved in the biotransformation (both phase I and phase II) of a wide range of substrates such as lipids, carbohydrates, and proteins. These biotransformation genes included those encoding cytochrome P450 (CYP), glutathione S-transferases (GSTs), UDP-glucuronosyltransferases (UGT), NADPH-cytochrome P450 reductase homolog (emb-8), and a number of genes annotated as FAD/NADP coenzymes. Cytochrome genes cyp-25A1, cyp-25A2, cyp-29A2, cyp-33B1, cyp-35B1, and cyp-37A1 were upregulated in all wastewater samples. Transcriptional repression was found for pathways involved in the metabolism of purine and pyrimidine nucleotides and was identified in nematodes exposed to samples C and H. We also found DEGs involved in a peroxisomal pathway, including the transcripts of acox-3, prx-3, prx-5, gstk-1, daf-22, ctl-2, ech-4, fard-1, acs-13, C24A3.4, T20B3.1, and ZK550.6 genes upregulated by samples C and prx-3, C24A3.4, daao-1, prx-14, ctl-2, ech-4, sod-1, and acs-13 upregulated by sample N. Genes annotated for oxidative stress response were found upregulated, including pdi-2 and F09F3.5 (in sample C), pept-1 (in N), R08F11.7 (in C and N), and col-61 .Gene ontology (GO) and domain enrichment analysis of DEG lists were carried out in DAVID software to identify the types of biological mechanisms underlying the nematode responses triggered by exposure to wastewater samples  of all DEGs, which responded to the wastewater samples, revealed several transcripts that can be linked to reproductive physiological processes in C. elegans (Table S4). Nevertheless, reproduction-related processes (GO:0000003) were not found among the significantly regulated processes as obtained by GO enrichment analysis. We also found in total 40 DEGs encoding C-type lectin (CLEC) proteins, which are related to the immune response in nematodes. Of these, 11 genes were differentially expressed in all wastewater samples including both upregulation  and downregulation .Also genes involved in the To validate the microarray results, we conducted RT-qPCR for 15 target genes that were among the top most affected transcripts, among those regulated in all wastewater samples, or those specifically responding to one or two wastewater samples. Overall, RT-qPCR results correlated to the microarray results Fig. .Fig. 7VaCaenorhabditis elegans to fingerprint wastewaters before and after treatment by a WWTP and effluent receiving surface waters. Several genes were differentially regulated following the exposure to wastewater samples, and this effect was absent in nematodes exposed to treated effluent as well as in effluent receiving surface water. The nematodes were exposed without extraction or preconcentration of water samples, except the removal of suspended solid materials by centrifugation. This means that bioanalysis with the water-exposed nematodes will especially indicate the total toxic potencies of bioactive pollutants (including hydrophilic compounds) that may be present in the tested samples, even at concentrations that could not yet be detected with chemical analysis.In this study, we successfully applied a nematode-based assay using gene expression profiling in Untreated and treated wastewater can typically contain a wide range of natural and synthetic chemical contaminants and reaction products and metabolites thereof  in all four types of wastewaters, these sample types also had specific DEGs that could be characteristic for the source. These included 31%, 4%, 35%, and 13% of the total DEGs specifically regulated in response to the sample C, H, N, and I exposure, respectively. There were also several DEGs regulated in the nematodes treated with the samples C, H, and N, but were not found in the sample I exposure. Compared with the total amount of DEGs found with each wastewater source, these genes comprised 74% for C-affected, 35% for H-affected, and 83% for N-affected DEGs (including the overlaps). The expression of these genes may be linked to substances that were diluted by the additional water from other sources  such as stormwater runoff, seepage water, and water from other community households. It is also possible that the substances in wastewater sources were degraded or have reacted before reaching the influent. More detailed study, including more sampling (time) points and combining this with a tiered approach for screening and assessment of the contaminant mixtures, can reveal the most important bioactive compounds, their sources, and their fate. This is comparable to the approach of effect-directed analysis (EDA) utilizing the process similar to the toxicity identification evaluation (TIE) to identify unknown contributors to the mixture effects in water samples as described previously by Escher et al. .Only two genes were regulated in the nematodes treated with effluent, suggesting an efficient removal of bioactive pollutants by the WWTP, and none after emission of the effluent into the surface water. This means that the nematode assay could be developed into a bioanalytical tool for determining whether the toxic potency is below a threshold of ‘no indications for concern’. The small size of the nematodes and sensitivity of molecular endpoints potentially make the assay sensitive for ultra-low concentrations of contaminants. The aim, however, does not necessarily have to be to make the assay as sensitive as possible, but sensitive enough to be able to determine whether the possibly remaining contaminants do not pose a risk.emb-8 gene encoding C. elegans NADPH-cytochrome P450 reductase homolog (EMB-8) which governs the nematode CYP-mediated metabolism  proteins. This could be related to exposure of the nematodes to microorganisms from the wastewaters including pathogens that may trigger an immune response in the nematodes as previously reported by Irazoqui et al. . ProteinC. elegans is a potential powerful tool for monitoring water-soluble pollutants in wastewaters. This bioanalytical assay especially is suitable for monitoring of the mechanism-specific toxic potency from bioactive pollutants (including hydrophilic compounds) since the nematodes can be directly exposed to even severely polluted wastewater samples without the need to pretreat or to dilute the samples. The results from this study showed a strong difference between polluted water and clean(ed) water samples in terms of gene expression profiles and intensity. Hence, our method can be used for monitoring the removal efficiency of (micro)pollutants during wastewater treatment and assessing the quality of the resulting effluent and receiving waters. In a tiered approach, this bioanalytical tool could help identify the most important bioactive compounds, their sources, and their fate. Also, the mechanistic profile of specific compounds of interest could be studied to possibly be able to identify, for instance, the presence of  drugs in wastewater. In addition, transcriptional profiles could be used to identify the presence of wastewater input or specific wastewater sources. It also is important to study the lowest induction level below which there is no indication for toxicological concern from hydrophilic compounds, compounds that are not yet easily detected, quantified, and assessed based on chemical analysis.Overall, this study showed that gene expression profiling in Supplementary file1 (XLSX 70 kb)Below is the link to the electronic supplementary material.


Due to the low incidence and the large number of postmortem diagnoses, treatment recommendations for intravascular large B-cell lymphoma (IVLBCL) are largely based on retrospective studies and case reports. There is little data on autologous stem cell transplantation (ASCT) in dialysis-dependent patients and choosing an adequate regimen and dosing is difficult. Here, we report the treatment of a patient with relapsed IVLBCL and end-stage renal disease caused by lymphoma-associated renal AA amyloidosis using a modified TEAM  regimen and ASCT. A 42-year-old female had an early relapse of hemophagocytic syndrome-associated intravascular large B-cell lymphoma resulting in terminal renal disease with dialysis dependency. Because of comorbidities , a modified, dose-reduced TEAM regimen was used as a high-dose conditioning regimen based on clinical pharmacologic considerations. The patient developed grade three mucositis and grade four febrile neutropenia as adverse events after transplantation. A modified TEAM regimen is feasible in a patient with end-stage renal disease with manageable toxicity. This is the first report of treatment with thiotepa in a dialysis-dependent patient. Intravascular large B-cell lymphoma (IVLBCL) is a rare disease characterized by the proliferation of lymphoma cells within the lumina of small vessels. Even though early diagnosis and treatment are vital, the diagnosis is often delayed due to a variety of possible symptoms and the lack of detectable tumor masses. The new WHO classification for neoplasms and lymphomas distinguishes between two variants of IVLBCL : the claTreating a rare disease like intravascular lymphoma is challenging. Due to the low incidence and large number of postmortem diagnoses, treatment recommendations for IVLBCL are largely based on retrospective studies and case reports. Standard R-CHOP achieves a complete remission rate of up to 90%, but the relapse rate is very high with a two-year progression-free survival (PFS) of only 56% -4. A recHere, we report the treatment of a patient with relapsed IVLBCL and end-stage renal disease caused by lymphoma-associated renal AA amyloidosis using a modified TEAM  regimen and ASCT.This article was previously presented as a meeting abstract at the 2020 DGHO Meeting on October 09, 2020.2 over four weeks) was begun [A 42-year-old female presented with fever, hepatosplenomegaly, and pancytopenia. She was diagnosed with hemophagocytic lymphohistiocytosis (HLH) due to fulfilling six , and hemophagocytosis) of the eight diagnostic criteria . Extensias begun . No clinas begun . The pat2 on Day 0, gemcitabine 1000 mg/m2 on Day 2 and Day 9, dexamethasone 40 mg on Day 1 to 4, and cisplatin 75 mg/m2 on Day 1) for five cycles resulting in a partial response. Stem cells were successfully harvested after the third cycle. Because of comorbidities , a modified, dose-reduced TEAM regimen was used as a high-dose conditioning regimen based on clinical pharmacologic considerations. The TEAM regimen consisted of thiotepa 5 mg/kg on Day -7, etoposide 100 mg/m2, and cytarabine 200 mg/m2 once daily on Days -6 to -3, and melphalan 100 mg/m2 on Day -2, as seen in Table As salvage treatment, she received R-GDP , the main and active metabolite of thiotepa with similar alkylating properties, was increased by about 150% with the split application over four days. Exposure of other metabolites  was not quantified . The phaThe hematological recovery after ASCT was delayed, which could be caused by prolonged fever after transplantation or increased toxicity by overdosing. We could not confirm relapse before death but had a strong suspicion due to elevated serum sIL-2R.The presented case illustrates that a modified TEAM regimen is feasible in a patient with end-stage renal disease with manageable toxicity. To our knowledge, this is the first report of thiotepa in a dialysis-dependent patient. Due to the rapidly progressive and fatal course of IVLBCL, it is essential to aim for aggressive, i.e. blind multiorgan biopsy particularly in patients, where HLH-associated lymphoma is suspected. Early, aggressive treatment with R-CHOP or R-CHOEP and high-dose chemotherapy with ASCT should be considered in high-risk and eligible patients.


In recent years, a growing body of research has revealed that long noncoding RNAs (lncRNAs) participate in regulating genomic instability.  We obtained RNA expression profiles, somatic mutation profiles, clinical information, and pathological features of colorectal cancer (CRC) from The Cancer Genome Atlas project. We divided the cohort into two groups based on mutation frequency and identified genomic instability-related lncRNAs (GI-lncRNAs) using R software. We further analyzed the function of identified GI-lncRNAs and established a prognostic model through Cox regression. Using the established prognostic model, we divided the cohort into the high- and low-risk groups and further verified the prognostic differences between the two groups as well as the predictive power of prognosis-related lncRNAs in the genomic instability of CRC.  We identified a total of 143 GI-lncRNAs that were differentially expressed between the higher mutation frequency group and the lower mutation frequency group. According to Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology analyses, a series of cancer-associated terms were enriched. We further constructed a prognostic model that included five GI-lncRNAs . We confirmed that the expression of the five GI-lncRNAs was associated with prognosis and the mutation of critical genes in the CRC patient cohort.  The present research further confirmed the vital function of GI-lncRNAs in the genomic instability of CRC. The five GI-lncRNAs identified in our study are potential biomarkers and need to be studied in more depth. Colorectal cancer (CRC) is the most common malignant neoplasm of the digestive system. According to “Cancer Statistics, 2020” produced by the American Cancer Society, a total of 147,950 new cases and 53,200 deaths were registered from CRC in the United States [Genomic instability, including chromosomal instability and microsatellite instability (MSI), is an immensely complex molecular phenotype and mechanism . GenomicWith the development and utilization of next-generation sequencing platforms, an increasing number of novel genes and mutations have been identified and verified to play critical roles in tumor progression. For example, exome sequencing was utilized to identify CRC somatic mutations, and paternally expressed gene 3 (PEG3) was verified as a high-frequency mutated gene . Imperiain silico data from The Cancer Genome Atlas  project [Recently, an accumulating body of bioinformatic studies was focused on the identification of potential biomarkers associated with tumor progression. For instance, seven identified miRNAs  were found related to development, lifestyles, and overall survival of breast cancer patients . Lu et a project  and perfhttps://portal.gdc.cancer.gov). The inclusion criteria were as follows: the samples (1) were pathologically diagnosed as colon adenocarcinoma or rectal adenocarcinoma, (2) had integral clinical and pathological data, and (3) had the follow-up time and surviving state. As a result, a total of 568 CRC and 44 normal tissues were included in this research. According to the documents and guidelines published by TCGA Authority (https://cancergenome.nih.gov/publications/publicationguidelines), the present research does not require ethical review.We downloaded transcriptome profiles (RNA-Seq), simple nucleotide variation (masked somatic mutation), and clinical data from TCGA database  and a genomically unstable-like group (GU-like group) through clustering analysis. Genetic mutations and gene expressions between the two groups were also analyzed.We extracted the lncRNA expression matrix and genetic mutation information from TCGA samples using PERL software. The samples were then ranked according to the number of cumulative mutant genes. We further selected the top 25% and bottom 25% of the samples to compare differences in lncRNA expression. GI-lncRNAs were identified using the “edgeR” package in R software  with a tP < 0.05 was regarded as statistically significant.We performed Spearman's correlation analysis to identify the mRNAs that were coexpressed with GI-lncRNAs. Each lncRNA that corresponded to 10 mRNAs was shown in a network diagram. Moreover, we performed Gene Ontology (GO)  and KyotP < 0.05). Calculation of hazard ratio and 95% confidence interval and generation of the profile of the receiver operating characteristic (ROC) curve were performed by applying “survival,” “caret,” “glmnet,” “survminer,” and “timeROC” packages in R software.A total of 509 CRC patients, whose follow-up time was more than 30 days and for whom complete clinical data were available, were enrolled in this study. We randomly divided the samples into a “train” group and a “test” group, and then, we performed Cox proportional hazard regression analysis to identify the GI-lncRNA signature. We then identified GI-lncRNAs which were significantly related to survival in the “train” group and the “test” group  and the GU samples (n = 128). A total of 143 GI-lncRNAs were identified which were significantly differentially expressed between the two groups. Among the GI-lncRNAs, 67 GI-lncRNAs were upregulated and 76 GI-lncRNAs were downregulated. The list of GI-lncRNAs is shown in Supplementary Materials, and the top 20 upregulated and downregulated GI-lncRNAs are shown as a heatmap in To identify the GI-lncRNAs in CRC, we compared lncRNA expression between the GS samples (P < 2.22e − 16). Moreover, we analyzed differences in the expression of mismatch repair genes and colorectal oncogenes between the two groups. The expression of caudal-related homeobox transcription factor 2 (CDX2) (P < 2.22e − 16), mismatch repair gene mutL homolog 1 (MLH1) (P = 8.3e − 10), and postmeiotic segregation increased 2 (PMS2) (P = 0.014) was significantly lower in the GU-like group, while expression of epidermal growth factor receptor (EGFR) (P = 1.2e − 4) was higher in the GU-like group. However, there was no significant difference in the expression of mutator S homolog 2 (MSH2) (P = 0.7) or mutator S homolog 6 (MSH6) (P = 0.066) between the two groups.We performed cluster detection to regroup the 568 samples into a GS-like group and a GU-like group. As a result, 363 samples were classified into the GS-like group, and 203 samples were classified into the GU-like group. The differential expression of GI-lncRNAs between the two groups is shown as a heatmap in Supplementary Figure 2 (MSH2) , P = 0.7To further explore the potential function of the 143 GI-lncRNAs, we performed GO and KEGG pathway analyses using R software. As shown in Supplementary Materials, we analyzed the 10 mRNAs with the highest coexpression coefficient with GI-lncRNAs. Moreover, we constructed the coexpression network that included the GI-lncRNAs and mRNAs . Moreover, with the increasing risk score, tumor somatic mutation count also increased, especially ranking between 50 and 150 . The above results confirmed the consistency and robustness of our model.We further utilized the “test” group samples and all TCGA set samples to verify the accuracy and reliability of the GI-lncRNA signature. The ROC curve analysis of the “test” set and TCGA set yielded AUCs of 0.658 and 0.704, respectively Figures . We alsoP < 0.001, and P = 0.036). Similarly, the high-risk group had lower overall survival in both the female and the male sets . Moreover, we stratified the samples into T1-2 and T3-4 sets, N0 and N1–3 sets, and M0 and M1 sets. Kaplan-Meier analyses indicated that the high-risk group had a poorer prognosis than the low-risk group in the T1-2 and T3-4 set . We also observed similar results in the N0, N1–3, and M0 sets . However, there was no significant difference in the M1 set (P = 0.254).We next calculated the hazard ratio and 95% confidence interval of age, TNM stage, and risk score in TCGA set. As shown in To compare the present prognostic model with the existing lncRNA-related signature, we further performed ROC curve analysis using the same TCGA cohort. Previously published research included a five-lncRNA signature derived from Gu et al. , six-lncP = 0.008 and P = 0.014). However, PIK3CA  mutation manifested a higher frequency of mutation in the low-risk group (P = 0.006). The mutation of KRAS showed no difference between the two groups (P = 0.708). To a certain extent, the results indicated that the GI-lncRNA signature was able to predict the mutation of BRAF, TP53, and PIK3CA.We further analyzed whether the GI-lncRNA signature could predict CRC genetic mutation. We divided all samples into the high-risk and low-risk groups based on the median risk score. The results are shown in Noncoding RNAs including circRNAs , 25, miRIn the present study, we conducted bioinformatic analysis and identified a total of 143 GI-lncRNAs. However, there is still little research into the role of GI-lncRNAs in CRC. We performed cluster detection to regroup the samples into a GU-like group and a GS-like group. We found that CDX2, MLH1, and PMS2 were expressed at significantly lower levels in the GU-like group. CDX2 has been verified to be a critical biomarker of normal epithelium and prognosis in CRC patients , 35. FurWe conducted GO and KEGG analyses to uncover the biological function of GI-lncRNAs. The results revealed multiple enriched terms related to immunoregulation, genomic instability, and chemokine activity. For instance, incubation of colon cells with IL-6 (interleukin-6) engendered migration of MSH3 from the nucleus to the cytosol and promoted MSI . WunderlFurthermore, we performed Cox proportional hazard regression and identified a five-GI-lncRNA signature . PTPRD-AS1 has been identified as an immune-related biomarker which predicts overall survival and immunotherapeutic response in bladder cancer . AC00923A wide array of studies has demonstrated that genetic mutation influences the drug sensitivity and biological behavior of tumors . In CRC,The present research is mainly based on bioinformatic analysis and still has some limitations. Firstly, chromosomal instability and MSI have been revealed to have a critical role in genomic instability, but the simple nucleotide variation data could only supply information on mutant genes. Secondly, more molecular biological experiments are needed to verify the identified biomarkers and mechanisms involved in the future.Our study provided a bioinformatic strategy to identify lncRNAs and potential mechanisms based on TCGA database and bioinformatic software. Moreover, we identified the five-GI-lncRNA signature as an independent prognostic marker in different cohorts. This GI-lncRNA signature has profound significance in genomic instability and certain value for further research.


Gastric adenocarcinoma (GAD) is one of the most common tumors in the world and the prognosis is still very poor.  We sought to identify reliable prognostic biomarkers for the progression of GAD and the sensitivity to drug therapy.  The RNA sequencing data of GAD was downloaded from the Cancer Genome Atlas (TCGA) database and used for analysis. Differentially expressed, immune-related lncRNA (DEIRlncRNA) was characterized by differential analysis and correlation analysis. Univariate Cox regression analysis was used to identify DEIRlncRNA associated with prognosis. Least absolute shrinkage and selection operator (LASSO) regression analysis allowed us to determine a signature composed of eight IRlncRNAs. Based on this signature, we further performed gene set enrichment analysis (GSEA) and somatic mutation analysis to evaluate the ability of this signature to predict prognosis.  In total, 72 immune-related lncRNAs (DEIRlncRNAs) with prognostic value were identified. These lncRNAs were used to construct a model containing eight immune-related lncRNAs (8-IRlncRNAs). Based on this risk model, we divided GAD patients into high-risk and low-risk groups. The analysis showed that the prognosis of the two groups was different and that the high-risk group had worse overall survival (OS). Immune cell infiltration analysis showed that the proportion of memory B cells increased in the high-risk group while the proportion of macrophages M1, T cells, CD4 memory-activated cells, and T cell follicular helpers decreased. GSEA results showed that 8-IRlncRNA was significantly enriched in tumorigenesis pathways such as myc. The results of somatic mutation analysis showed that the CDH1 gene was significantly mutated in the high-risk group.  A prognostic signature of 8-IRlncRNAs in GAD was established and this signature was able to predict the prognosis of GAD patients. Gastric cancer (GC) has been identified as the fifth most common cancer and the third most common cause of death in the world . Due to 2+-triggered signal transduction [+ T cells and promotes the progression of GC [+, CD8+, and CD45RO+ cells is closely related to the survival of GC patients and regional lymph node metastasis [LncRNA are noncoding RNAs with a length greater than 200 nucleotides and have been shown to be involved in the progression of GC. LncRNA PVT1 promotes GC tumor growth and metastasis by stabilizing FOXM1 protein . LncRNA sduction . The proon of GC . The higtastasis . In additastasis , 17. Protastasis , 19. Howtastasis . AlthougBased on this, our study analyzed the RNA sequencing data of GAD obtained from TCGA and immune-related mRNA obtained from the Molecular Signatures Database. We established an 8-immune-related lncRNA (8-IRlncRNA) signature by LASSO Cox regression analysis. GSEA was used to explore immune-related response pathways. Furthermore, we evaluated the predictive potential of this signature from the perspective of immune cell infiltration and ICIs.https://portal.gdc.cancer.gov/). The inclusion criteria for the samples in this study were as follows: (1) samples included complete clinicopathological information; (2) samples with an OS time of less than 100 days were excluded. Using these criteria we selected a total of 279 patients including 24 normal samples and 279 cancer samples. The clinicopathological characteristics of the included patients are summarized in https://www.broadinstitute.org/gsea/msigdb/index.jsp: Immune System Processes M13664, Immune Responses M19817, v4.0).The RNA sequencing and clinical information data of GAD was downloaded from the TCGA database  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. GO analysis included biological process (BP), cell composition (CC), and molecular function (MF). The cutoff criterion was P value <0.05.We used the “limma” package in R software (v4.0.4) to perform differentially expressed analysis on GAD samples and normal gastric samples to identify differentially expressed mRNA (DEIRmRNA) and differentially expressed lncRNA (DElncRNA). The selection criteria for DEIRmRNA and DElncRNA were a R| > 0.4 and P value <0.05 were set as cutoff criteria.In order to further identify differentially expressed immune-related lncRNA (DEIRlncRNA), we carried out Pearson correlation analysis between DElncRNA and DEIRmRNA. Pearson correlation coefficient |n = 195) and test set (n = 84). The training set was used to explore and construct an IRlncRNA signature. The test set and entire GAD patient cohort (n = 279) were used to verify the IRlncRNA signature. Finally, we used these IRlncRNAs in multivariate Cox regression to obtain coefficients and determine 8 IRlncRNAs (8-IRlncRNA) that were significantly related to prognosis. Thereby, a predictive model weighted by their coefficients was established. We constructed a risk score formula for OS to assign a risk score to each patient:i means the coefficients and xi is the counts value of each lncRNA. Risk scores were computed for all patients included in our study. The low-risk group and the high-risk group were determined by the best cutoff value. Best cutoff value referred to the risk score with the largest difference in survival between the two groups at the lowest log rank P value.We used the “survival” R package to perform univariate Cox regression analysis on the selected DEIRlncRNA to further select the IRlncRNA related to the prognosis of GAD patients. Second, we used LASSO regression analysis to identify a meaningful prognostic signature. As mentioned earlier, a total of 279 GAD samples were used to construct IRlncRNA signature. We randomly divided the 279 GAD samples into training set (n = 84) and the entire GAD cohort (n = 279). The low-risk group and high-risk group were determined by the best cutoff value. A P value <0.05 was considered statistically significant.We used the “timeROC” R package to perform the time-varying receiver operating characteristic (ROC) curve analysis in the training set and draw a Kaplan–Meier (KM) survival curve to compare the OS difference between the high-risk group and the low-risk group for evaluating the predictive value of the 8-IRlncRNA signature. Similarly, the stability and reliability of the 8-IRlncRNA signature were verified through the above analysis in the test set  was used in the analysis. The predefined gene set of “hallmark.v6.2.symbols.gmt” was downloaded from the molecular marker database. The false discovery rate (FDR) < 0.25 was defined as the critical value and sorted by normalized enrichment score (NES) value.Gene set enrichment analysis (GSEA) is a method used to determine if a predefined gene set can show significant consistent differences in two biological states. Therefore, we performed a GSEA to explore the potential function of the 8-IRlncRNA prognostic signature. The low-risk group and the high-risk group were determined according to the best cutoff value and GSEA software  were identified. We performed univariate Cox regression analysis on these 648 DEIRlncRNAs for the purpose of selecting IRlncRNAs related to prognosis in GAD patients. A total of 94 prognostically related IRlncRNAs were identified. Then, we used LASSO  and multTo evaluate the prognostic ability of the 8-IRlncRNA signature in GAD and assign a risk score to each patient, we determined the risk score of the 8-IRlncRNA signature. This risk score is based on the expression of these 8 IRlncRNAs and their ability to predict OS. The specific formula is: Risk score = 0.0105512600 × (RP11-497E19.1) + 0.0147859892 × (CH17-118O6.3) + 0.0004966951 × (RP11-54A9.1) + 0.0035751428 × (RP11-1260E13.4) − 0.0008728424 × (RP11-489D6.2) + 0.0014697293 × (AC093850.2) + 0.0081959700 × (AC010890.1) + 0.0003624435 × (RP11-115H13.1). According to the best cutoff value, patients were divided into high-risk groups and low-risk groups . Seven oIn the training set, the mortality rate of the high-risk group was higher than that of the low-risk group . The expWe also used the above formula to calculate the risk score of the 8-lncRNA signature to verify the predictive ability on OS. Then, the signature was verified in the test set and the entire GAD cohort  were included in this part of the analysis. Among the molecular subtypes, the genomically stable (GS) subtype had the highest risk score, while the Epstein–Barr virus (EBV) subtype had the lowest risk score. Meanwhile, the risk scores of chromosomal instability (CIN) and microsatellite instability (MSI) subtypes were between GS and EBV . The anaWe performed GSEA on the high-risk and low-risk groups to uncover the possible molecular mechanisms of 8-IRlncRNA prognostic signatures in GAD. The results show that the low-risk group was mainly enriched in myc targets and DNA repair Figures . The higP < 0.0001)  in both the high-risk group and the low-risk group, especially in the TTN and TP53 genes Figures . The mut Figures . The resWe analyzed the abundance of 22 tumor-infiltrating immune cells in the high-risk and low-risk groups. Compared with the low-risk group, the ratio of B memory cells in the high-risk group increased, while the ratio of macrophages M1, T cells CD4 memory activated, and T follicular helper cells decreased . These rICI, as a kind of tumor immunotherapy, is a promising treatment method for GC. Immune checkpoint molecules include PD-1, PD-L1, cytotoxic T lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin mucin-3 (TIM-3), and others . We explGC has the characteristics of high incidence and low 5-year survival rate , 27. It β-catenin, which suggests that AC093850.2 can promote gastric cancer cell proliferation and invasion by activating Wnt/β-catenin signaling [We identified seven IRlncRNAs  that were associated with high risk. Only one IRlncRNA (RP11-489D6.2) was associated with low risk. According to the risk scores generated by the expression levels of these 8 IRlncRNAs, we divided GAD patients into a low-risk and a high-risk group. Our results showed that the prognosis of the two groups were different, and the high-risk group had a poorer OS. The AUC values of 1-year, 3-year, and 5-year OS were higher in the training set, validation set, and all gastric adenocarcinoma patient cohorts. These results suggest that this 8-IRlncRNA signature has the potential to differentiate the prognosis of GAD patients. Among these 8 IRlncRNAs, knockout of AC093850.2 (LINC00460) inhibits tumorigenesis in vivo and the growth and invasion of cervical cancer cells in vitro. These studies show that AC093850.2 plays a carcinogenic role in cervical cancer . Overexpignaling , 33. In ignaling . In addiignaling . Subsequignaling . Our anaignaling . It has ignaling . In our + B cells may increase the risk of GC development; once cancer develops, suppression of IL-10+ B cells may enhance overall antitumor immunity [The tumor immune microenvironment is of great value in prognostic research on GC . In ordeimmunity . Immunotimmunity . ICI, a immunity . Immune immunity . In our immunity , 47. Twoimmunity , 48. Wanimmunity . The uprimmunity , 51. Koyimmunity . Anti-TIimmunity . Our resThis is the first study to identify an 8-IRlncRNA signature with good potency, including in predicting prognosis and response to immunotherapy, and also it is one of the few studies that combines the identification of IRlncRNA signature with somatic mutation analysis. The limitations of our study are as follows: there is lack of a suitable external dataset to validate our model, necessitating the use of data obtained from the TCGA database; and some of the lncRNAs in our identified signatures have not been characterized, and so additional in vitro and in vivo experiments are needed.Gastric adenocarcinoma (GAD) is one of the most common tumors in the world and the prognosis remains very poor. It is necessary to develop biomarkers that can be used to predict the prognosis of gastric cancer. We established a prognostic signature of 8-IRlncRNA in GAD based on the LASSO model and verified that this signature can be used as a prognostic marker for GAD. We also explored the relationship between this prognostic signature and tumor immune characteristics, and the role of the 8-IRlncRNA prognostic signature in predicting immunotherapy response. However, the application value of this signature needs further validation so as to provide reliable evidence for clinical use.


Age and sex-stratified comparison revealed increased susceptibility to COVID-19 only from females with DM. For either non-DM or DM group, hyperglycaemia was associated with adverse outcomes, featured by higher rates of severe pneumonia and mortality, in comparison with non-hyperglycaemia. This was accompanied by significantly altered laboratory indicators including lymphocyte and neutrophil percentage, C-reactive protein and urea nitrogen level, all with correlation coefficients >0.35. Both diabetes and hyperglycaemia were independently associated with adverse prognosis of COVID-19, with hazard ratios of 10.41 and 3.58, respectively.This study is performed to figure out how the presence of diabetes affects the infection, progression and prognosis of 2019 novel coronavirus disease (COVID-19), and the effective therapy that can treat the diabetes-complicated patients with COVID-19. A multicentre study was performed in four hospitals. COVID-19 patients with diabetes mellitus (DM) or hyperglycaemia were compared with those without these conditions and matched by propensity score matching for their clinical progress and outcome. Totally, 2444 confirmed COVID-19 patients were recruited, from whom 336 had DM. Compared to 1344 non-DM patients with age and sex matched, DM-COVID-19 patients had significantly higher rates of intensive care unit entrance (12.43%  The World Health Organization recently declared the outbreak of 2019 novel coronavirus pneumonia (COVID-19) a global pandemic. Increased morbidity and mortality are particularly seen in older persons and those presenting with comorbidities such as overt diabetes, obesity and hypertension . Among aHowever, a high degree of heterogeneity is observed in diabetes population, such as type of diabetes, quality of glucose control, duration of disease as well as diabetic complications all show diversity. One key question is the effect on poorer outcomes because of DM or the hyperglycaemia cannot be separated, as COVID-19 can cause secondary hyperglycaemia as revealed in previous studies . The mulA multicentre study was performed in four hospitals in Wuhan between 1st January and 31st March 2020, i.e. Tianyou Hospital of Wuhan University of Science and Technology, Wuhan First Hospital, Renmin Hospital of Wuhan University and Tongji Hospital, Tongji Medical College. All are the major tertiary hospitals in Wuhan and responsible for the treatments for COVID-19 assigned by the government. COVID-19 was diagnosed as positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by real-time reverse transcriptase-polymerase chain reaction assay of oropharyngeal swab specimens . This stWe defined illness onset as the first day of reported symptoms consistent with COVID-19. According to ADA's criteria for the diagnosis of diabetes , patients.d) if they are normally distributed or median  if not. Categorical variables between groups were analysed using Student's t test or Mann–Whitney U test as appropriate. Mean values and percentages were compared among different groups by analysis of variance and χ2 tests, as appropriate. For comparison of clinical symptoms, laboratory measurements and clinical outcomes between DM and non-DM, we applied propensity score matching (PSM) to adjust sex and age using the nearest-neighbour matching method by R package ‘MatchIt’. PSM refers to the expression of the influence of multiple covariables by the change of propensity score (PS) value, matching between groups according to PS value and finally analysing the effect value within the matching group with balanced distribution of covariables [P value of <0.05 was considered statistically significant.Categorical variables were expressed as frequency and percentages (%), and continuous variables were expressed as mean , DM with coronary heart disease (CHD) (n = 52) and DM with cerebrovascular disease (CVD) (n = 26). Older age (65 vs. 60) rather than sex  was associated with the presence of DM  . Of these 2444 patients, 35.72% were men and 35.9% were >65 years old. The median duration from symptoms onset to hospital admission and from hospitalisation to death were 8  and 7  days, respectively. Based on criteria, 336 patients had DM, including DM with hypertension (ce of DM . The com< 0.001) .Table 1vs. 25.0% vs. 9.5%, P = 0.032). However, due to the small sample size, we did not find significant differences in other characteristics and clinical outcomes among the three groups (Supplementary Table S4).For patients without a history of DM, we redefined DM in terms of HbA1c levels . HbA1c was measured in a total of 99 patients, 66 of whom had no history of DM. Of the 66 patients, 21 was classified as normal group, 20 had pre-DM and 25 had DM. DM patients were more likely to have hyperglycaemia and pre-DM patients also had a higher risk of hyperglycaemia than those with normal HbA1c , this overrepresentation was also observed in the 18–29, 40–49 and ≥70 years old, and for male and female separately. In contrast, a highly comparable proportion of DM was observed between COVID-19 patients and that of the general population (13.7% vs. 12.8%), and a lower proportion was observed in COVID-19 patients of 50–59, 60–69 and ≥70 age groups. More DM was found in the female while less DM was found in the male for the COVID-19 patients than the general population . The number of laboratory evaluations that was significantly different between two groups was reduced to nine (Supplementary Table S1). As expected, the DM patients had higher levels of random plasma glucose (8.02 vs. 5.69) and HbA1c (8 vs. 5.9) (Supplementary Table S1).To attain comparability between the DM and non-DM patients, we selected 1344 non-DM patients with age and sex matched by PSM. The coexistence of other medical conditions was still higher in the DM group than the non-DM, including more hypertension and CHD . Significantly higher mortality rate was observed from the DM patients than the matched non-DM COVID-19 patients   .N = 3), and DM–coronary disease (N = 8) were not further analysed due to the small sample size.We made additional analysis on the COVID-19 patients with isolated DM and those with other comorbidities apart from DM. The DM–hypertension as the most frequently seen coexisting condition, showing no significant effect on more severe COVID-19 disease or any more adverse outcomes, when compared with isolated DM COVID-19 patients, and after adjusting the effect from age and sex (Supplementary Table S2). Other coexisting conditions, including DM–CVD (P < 0.005), in comparison with those with non-hyperglycaemia . A significant negative interaction was observed between age and DM for the effect on death, with an HR of 0.95 , which indicated that the effect from simultaneous presence of older age and DM is lower than the sum of the isolated effect from older age and DM. This could be attributed to their overlapped effect on death. The delayed hospitalisation was related to mortality, with the HR < 1, indicating the shorter delay, the lower risk of fatal outcome. Prescription of glucocorticoid was also associated with significantly increased risk of death   .Table 3P < 0.001). The glucose level was also correlated with laboratory measurements that were indicative of liver function, renal function, inflammation responses and hypercoagulable state, which may contribute to a poorer prognosis of COVID-19, however, with the correlation coefficients less than 0.35 (Supplementary Table S3).To explore the laboratory indicators that might mediate the association between glucose and adverse clinical outcome, we made a correlation analysis between the longitudinal glucose level and the other commonly seen laboratory indicators. The glucose level was inversely correlated with the median value of lymphocyte percentage, while positively correlated with those of HbA1c, C-reactive protein (CRP), percentage of neutrophils and urea nitrogen, all with correlation coefficients over 0.35 with significance  [Diabetes has posed as an important public health problem in China, with highest risk outweighing that of all the other Asian countries . Consides. 7.1%) . A retros. 7.1%) . HoweverP < 0.001) [In China, the surveillance data of diabetes in the general population  have mad< 0.001) . Taken t< 0.001) , 34, hadvs. non-DM difference of the in-hospital laboratory abnormalities were identified during the hospitalisation that might be related to the adverse prognosis of DM-COVID-19, including elevated creatinine, urea nitrogen, uric acid and procalcitonin, four of them were indicative of kidney injury. This finding was consistent with the higher frequency of kidney failure in DM-COVID-19, a significant contributor to the fatal outcome. Multiple complications, especially renal disorders, were frequently encountered in DM patients [We also noted that when DM-COVID-19 patients were matched for age and sex, similar symptoms were observed as those without DM and their initial manifestation could be mild. However, significant DM patients . As has patients , which wA different panel of laboratory indictors was correlated with the glycaemic abnormalities, especially lymphopoenia, increased CRP, neutrophils percentage, all being inflammation-related biomarkers, indicating the degree of inflammation and possible secondary bacterial infection exacerbating COVID-19 , 36, 37.There are specific concerns as to the usage of steroids in patients with COVID-19. This remains controversial among numerous studies, although a very recent randomised controlled trial study provided evidence that dexamethasone is effective for severe patients with COVID-19 –42. In oIn summary, we found that both a known history of diabetes and glycaemic status during illness were independently predictive of COVID-19-related mortality. We were able to disclose that hyperglycaemia with or without DM during the hospitalisation were independent predictors for morbidity. Normalisation of blood glucose levels might be critical in reducing mortality and morbidity in both diabetic patients and non-diabetic patients. Intensive glucose monitoring and insulin therapy to obtain optimal metabolic control may improve the outcome of COVID-19 patients. These findings have the potential to provide therapeutic insights and contribute to precision medical interventions for COVID-19.


Cryoneurolysis, cold-induced reversible conduction block of peripheral nerves, is an effective treatment for reducing knee osteoarthritis (OA) symptoms and opioid use following knee arthroplasty. There are however, limited data concerning its use for ankle OA. Our aim was to assess clinically significant long-term symptomatic relief of ankle OA with cryoneurolysis.This single-center, open-label trial included participants aged >18 years with radiographic tibiotalar OA, unilateral ankle pain ≥5/10 on Numerical Rating Scale (NRS), and with no ankle surgery within 6-months of screening. Following ultrasound-guided cryoneurolysis of nerves in the participant's pain distribution , outcomes were assessed at clinic visits  and by telephone interview . The primary endpoint was change in Foot and Ankle Outcome Score (FAOS)  at 12-weeks. Change in quality of life (FAOS-QoL), activities of daily living (FAOS-ADL), NRS-pain, and physical performance measures were also assessed. Longitudinal mixed models were constructed to evaluate changes from baseline at 6, 12- and 24-weeks post-treatment.Forty participants enrolled . At 12-weeks post treatment, FAOS-pain , ADL , QoL  and NRS-pain  were significantly improved from baseline. No difference in 40-m fast-paced walking test was detected at 12-weeks post-treatment . For all outcomes, similar findings were observed at 6- and 24-week visits.Cryoneurolysis resulted in statistically significant improvements in ankle pain, physical function and QoL for up to 24-weeks in participants with unilateral, symptomatic ankle OA. In that study, a single cryoneurolysis treatment of the infrapatellar branches of the saphenous nerve resulted in a reduction in pain and symptoms for at least 90 days . Further22.1clinicaltrials.gov (NCT03567187). Participants were identified through hospital records, physician referrals, mass mailings, and advertisements.We conducted a single-center, single-arm open-label clinical trial with up to 24-weeks follow-up to examine the effectiveness of cryoneurolysis for reducing pain, improving activities of daily living (ADL) and quality of life (QoL) in patients with evidence of unilateral symptomatic ankle OA. We hypothesised that treatment of the superficial nerves and/or deep fibular nerve would result in significant improvements in function and pain at 24-weeks follow-up. This study was conducted at the University of Kansas Medical Center  from August 13, 2018 to January 26, 2021. Participants were assigned to undergo cryoneurolysis of either the Superficial Fibular Nerve (SFN), Sural Nerve (SN), and/or Saphenous Nerve, or the Deep Fibular Nerve (DFN) using ultrasound-guided cryoneurolysis. This study was performed in accordance with the provisions of the Declaration of Helsinki, and the protocol was approved by the University of Kansas Institutional Review Board (STUDY00142298). Written informed consent was obtained from all participants before inclusion. The trial was registered at 2.22, were ambulatory and able to comply with study procedures, had undergone at least one prior conservative OA treatment  and were willing to abstain from the use of protocol-restricted medications during the trial, and analgesics, other than acetaminophen, 1 week prior to the beginning of the trial.Eligible participants were men and women aged >18 years, had radiographic evidence of ankle OA (Kellgren-Lawrence (KL) Grade ≥2; measured on weight-bearing mortise views with 20° internal rotation), were limited by unilateral ankle pain rated on a Numerical Rating Scale (NRS) as ≥5/10 on most days over the last month, had a Foot and Ankle Outcome Score (FAOS)  of <75 , intra-articular (IA) corticosteroids within 3-months of screening, oral corticosteroids within 2-weeks of screening (unless on a chronic stable dose for ≥3 months prior to enrolment), women who were pregnant, ankle pain due to a condition other than OA, arthroscopy or open surgery of the ankle joint within 6 months of screening, planned/anticipated surgery of the index ankle during the 6-month trial period or had a diffuse pain condition . A full list of the exclusion criteria is included in 2.3All participants were followed for up to 24-weeks following cryoneurolysis. Demographic and clinical characteristics for all participants were captured at baseline, 3, 6, 9, 12, 18 and 24-weeks follow-up. ‘Responder’ status was assessed using the NRS for pain at clinic visits  and via web-form or telephone interviews (as per the participants preference) at 3-, 9- and 18-week post treatment. Non-responders were defined as reporting <20% pain relief with respect to their baseline NRS pain score. Participants who were identified as non-responders to initial treatment were eligible to receive cryoneurolysis of the other nerve group at the next clinic visit. For example, if a participant was a non-responder 9 weeks after superficial nerves were treated, then at the 12-week clinical follow-up the participant would be offered treatment of the deep fibular nerve and if received, the 12-week visit would then become the new baseline visit for assessing outcomes of the new treatment.2.4i)Superficial Fibular NerveDepending on the location of the participants ankle pain, either the superficial nerves  or the deep fibular nerve for deep ankle pain was treated with cryoneurolysis. A sonographically-guided diagnostic nerve block using 1% lidocaine with 1:100,000 epinephrine was performed for each nerve to confirm analgesia prior to treatment. Cutaneous sensation in the distribution of each nerve treated was assessed with a standardized nylon monofilament prior to and following cryoneurolysis. The anatomical locations of the respective nerves of interest and the cutaneous distribution tested are described below.ii)Sural NerveThe superficial fibular nerve (SFN) was sonographically located in the distal third of the leg at a location approximately 12 cm above the tip of the lateral malleolus, where it exited the fascia of the lateral compartment of the leg. Sensory function was tested on the dorsum of the foot.iii)Saphenous NerveThe sural nerve (SN) was sonographically located approximately 12 cm proximal to the posterior border of the lateral malleolus, and lateral to the Achilles’ tendon. Sensory function was tested on the lateral side of the ankle and fifth ray of the foot.iv)Deep Fibular NerveThe saphenous nerve was sonographically located approximately 7 cm proximal to the medial malleolus, and adjacent to the greater saphenous vein. Sensory function was tested just distal to the medial malleolus.The deep fibular nerve and anterior tibial artery were sonographically located by following the anterior surface of the tibia distally in transverse view. Generally, the deep fibular nerve was located lateral to the anterior tibial artery approximately 6 cm proximal to the ankle joint line. Sensory function was tested over the dorsum of the webspace between the first and second toes.2.52O) that is contained within the handpiece and no gas is injected into the body [The iovera° cryoneurolysis device  is approved by the United States Food and Drug Administration (510(k) clearances K133453 and K161835), and is used in surgical procedures to alter nerve function by forming precisely controlled, sub-dermal cold zones. Exposure to localised temperatures of −20 to −80 °C temporarily disrupts peripheral nerve function by axonal and myelin degeneration known as Wallerian degeneration ,27. Whenthe body .While the initial three participants were treated using a 6.9 mm 27G triple tip (model 307 between 8/13/2018–11/5/2018), most participants were treated with a 55 mm, 22-gauge single-point cryoprobe (model 155 between 12/3/2018–9/17/2020) due to the need for precise localization of the cryoprobe with continuous sonographic monitoring to avoid nerve damage. For each nerve treated, at least 4–8 cryoneurolysis cycles were completed .2.62) was calculated from body mass (kilograms) divided by the square of the participant's height in meters ; as measured by a trained member of the research team.At the screening/treatment visit, body mass index , from baseline to 6, 12, and 24 weeks post-treatment respectively, with the 12-week outcome being the  mm/day ,33. Base2.8I)Joint Symptoms, Activities of Daily Living and Quality of LifePain, ADL and QoL were assessed at baseline, 12 and 24 weeks post-treatment using the FAOS , a self-2.9Gait speed was measured in meters per second (m/sec) using the 40-m fast paced walking test (40 m FPWT). The 40m-FPWT is routinely used in clinical practice for the assessment of physical function and is one of the Osteoarthritis Research Society International (OARSI) recommended functional performance-based outcome measures for OA research .2.10Adverse events (AE) were systematically assessed throughout the study by phone and at each visit. In addition, participants were encouraged to contact the study team with information about AEs that occurred between each respective visit. When an AE was reported to the study staff, data including the type of event, onset/end dates, duration, severity, and outcome were collected and reported to the Principal Investigator (PI). The PI determined the severity of the event using the CTCAE version 5.0 guidelines . Followi2.11To assess the primary hypothesis that cryoneurolysis reduced ankle pain for at least 12 weeks in people with unilateral, symptomatic ankle OA, a sample size was estimated based on published effect sizes of 1.06 for FAOS-pain and FAOS-QoL, and 0.65 for FAOS-ADL . At an ea priori primary analysis examined the change in FAOS-Pain between baseline and 12 weeks following cryoneurolysis. The analyses of secondary  and tertiary (i.e. 40m-FPWT) outcomes involved longitudinal data collected at baseline, 6, 12, and 24 weeks. All response variables were modeled using longitudinal mixed models. Each model was based on two main effects: a random subject effect, and a fixed time effect consisting of four levels (baseline and three follow-up time points). The Akaike information criterion was used to determine an appropriate variance/covariance structure for each model. Each hypothesis was tested by examining appropriate contrasts and estimated linear forms in the overall mean and the main effects for time, using an alpha level of 0.05 to determine statistically significant change. All analyses were participant-based, with one ankle per participant, and were conducted using “PROC MIXED” in SAS Version 9.4  with results presented as least-square means (LS means) and 95% confidence intervals (95% CI).The 32. Most study participants localized their pain to the superficial nerve group. Five participants reported pain deep into the ankle joint for whom we performed deep fibular nerve treatment with cryoneurolysis. Thirty-one participants were initially treated with cryoneurolysis of superficial nerves  and 4 were initially treated at the deep fibular nerve. One participant who was initially treated at the deep fibular nerve was later treated in the superficial group and four participants who were initially treated with the superficial group later received a treatment of the deep fibular nerve. Baseline clinical and demographic characteristics of eligible participants are presented in Forty-three potentially eligible participants were screened. Of these, two were found by radiographs or weight-bearing CT (WBCT) scan to not have tibiotalar OA (KL = 0) and were excluded. In addition, one participant was excluded due to an unstable ankle that required bracing and opioid therapy. Subsequently, 40 participants with unilateral, painful ankle OA were recruited. Participants (50% women) had a mean ± standard deviation  age of 63.0  years and a BMI of 31.7  kg/mTwenty-two participants completed the full 24-week follow-up. Reasons for discontinuation are presented in 3.1For the primary endpoint, there was a statistically and clinically significant improvement in FAOS-Pain from baseline to 12-weeks post-treatment  of 20.8  points. Improvement was also detected in FAOS-Pain from baseline to 6- and 24-weeks, with 17.4  points and 21.7  points improvement respectively. For secondary outcomes, improvement was also found in FAOS-ADL, FAOS-QoL and NRS pain scores. For the FAOS-ADL subscale, a statistically significant improvement of 16.6  points was detected at 6-weeks, 18.1  points at 12-weeks and 20.1 points  at 24-weeks. For the FAOS-QoL subscale, statistically significant improvement of 22.4  points was observed at 6 weeks, 19.9  points at 12 weeks, and 25.4 points  at 24-weeks. Change in NRS pain score  for the prior 7 days was also improved from baseline at 6-weeks , 12-weeks  and 24-weeks  respectively. For the tertiary outcome, treatment was not associated with a statistically significant change in 40m-FPWT at any timepoint; see 3.2Over the 24-week follow-up period following cryoneurolysis, 19 participants reported a total of 42 adverse events. Of these, 2 were deemed to be unrelated to the intervention, 4 unlikely related, 18 possibly related, 14 probably related , and 5 definitely related . A full list of the reported adverse events is presented in 4This single-center, single-arm, open-label study is the first to evaluate the symptomatic and functional benefits of cryoneurolysis in participants with unilateral, symptomatic ankle OA. We observed statistically significant changes from baseline in FAOS subscales for pain, ADL, QoL, and in NRS-pain at 6, 12 and 24-weeks post-treatment. These data suggest that cryoneurolysis is likely to be effective to relieve pain and improve self-reported function in patients with symptomatic ankle OA, although walk time was not found to improve. These findings warrant further investigation and validation by future randomised, placebo-controlled trials.Cryoneurolysis has been shown to be an effective treatment for decreasing knee pain , and impIn the current study, cryoneurolysis was well tolerated by most participants with a total of 42 adverse events (AEs) reported in 19 participants over the full 24-week study period. The most common AEs were arthralgia (N = 14) and paraesthesia (N = 11), which were mostly mild in severity and did not require clinical intervention. The higher occurrence of these AEs in the initial group of participants was attributed to the use of the 6.9 mm sharp tips and was very rare after changing to 55 mm dull tips that allowed continuous sonographic guidance to avoid direct contact with the nerve. Even with sonographic guidance, it was thought that the use of the sharp tips may increase the risk of puncturing the nerve sheath leading to increased post-cryoneurolysis dysesthesias observed in the first three participants treated. After switching to the 55 mm tip, the procedure had substantially fewer AE's.When compared to a previous study of cryoneurolysis in patients with mild-to-moderate knee OA , we repoOne limitation of this study was that, upon re-assessment of the baseline radiographs following treatment, one participant had KL grade 4 talonavicular OA and one had subtalar joint OA, rather than tibiotalar OA. Given that these participants were treated for ankle pain and returned for follow-up, they were not excluded. Further, we used an open-label design without a sham treatment as this was the first study of cryoneurolysis for ankle OA, and the funding source elected to determine the magnitude of effect of the novel protocol used prior to initiating a controlled study. Large placebo effects have been observed in OA clinical trials, particularly in studies of surgical intervention . While i5Cryoneurolysis of the superficial and/or deep nerves surrounding the ankle resulted in significant improvements in ankle pain, function and QoL for up to 24-weeks in participants with unilateral, symptomatic ankle OA. These data support the use of cryoneurolysis as an effective and safe non-pharmacological treatment of joint symptoms in patients with symptomatic ankle OA.Funding for this study, equipment and cryoprobes were provided by Pacira CryoTech, Inc. (previously Myoscience) through an Investigator-Initiated Research (IIR) unrestricted grant. The funders were not involved in the study design, data collection and interpretation of study results.10.13039/100016220CTSA Award #UL1TR000001and 10.13039/100006093Patient-Centered Outcomes Research Institute (10.13039/100006093PCORI) Program Award #CDRN-1306-04631.Funding for this study, equipment and cryoprobes were provided by Pacira CryoTechh, Inc. (previously Myoscience) through an Investigator-Initiated Research (IIR) unrestricted grant. The sponsor was not involved in the study design, data collection, data analysis, data interpretation or manuscript preparation. HERON is supported in part by funds from Conception, design and conduct of study: NAS. Analysis and interpretation of data: both authors. Drafting Article: both authors. Critical revision of article: both authors. Final Approval: both authors.thomas.perry@kennedy.ox.ac.uk.All data generated and analysed in this study are available upon reasonable request. Access to data generated in this report should be sent to the corresponding author at PPI was not required nor involved with any aspect of the work presented.10.13039/100007859University of Kansas Institutional Review Board (STUDY00142298).Ethical approval was granted by the Neither author declares conflicts of interest related to the current study. NAS has consulted for Flexion Therapeutics for unrelated work.


Furthermore, there was a significant positive correlation between serum catestatin levels and vitamin D levels  while there was also a significant positive correlation between serum catestatin levels and growth differentiation factor-15 (GDF-15) levels . Furthermore, multivariate logistic regression showed that catestatin, GDF-15 and leukocyte count were significant predictors for COVID-19 survival. These findings imply that catestatin could be playing a major immunomodulatory role in the complex pathophysiology of the COVID-19 infection and that serum catestatin could also be a predictor of a poor COVID-19 outcome.Catestatin is a pleiotropic peptide with a wide range of immunomodulatory effects. Considering that patients with a severe COVID-19 infection have a major immunological dysregulation, the aim of this study was to evaluate catestatin levels in patients with COVID-19 treated in the intensive care unit (ICU) and to compare them between the fatal and non-fatal outcomes. The study included 152 patients with severe COVID-19, out of which 105 had a non-fatal outcome and 47 had a fatal outcome. Serum catestatin levels were estimated by an enzyme-linked immunosorbent assay in a commercially available diagnostic kit. The results show that catestatin levels were significantly lower in the fatal group compared to the non-fatal group (16.6 ± 7.8 vs. 23.2 ± 9.2 ng/mL;  Coronavirus disease 2019 (COVID-19) is a highly contagious infectious disease caused by the SARS-CoV-2 virus, responsible for over 3.8 million deaths worldwide. First reported in Wuhan, Hubei Province, China, in 2019, COVID-19 expanded to pandemic proportions in a short period of time, becoming the biggest world health crisis since 1918 and the influenza pandemic . MoreoveCatestatin is a pleiotropic peptide and one of the cleavage products of the pro-hormone chromogranin A. Its primary function is as an inhibitor of catecholamine secretion and stimulator of histamine release ,11. The As aforementioned, catestatin is known for its antimicrobial activity. Several studies investigated catestatin’s potential activity against bacteria, fungi and yeast ,25,26. SAlthough vitamin D was initially recognized as a regulator of calcium and bone metabolism, recent research has shown that vitamin D also exhibits a profound influence on the immune system . AccordiHence, the aim of this study was to investigate the serum levels of catestatin in ICU-treated COVID-19 patients. Moreover, we wanted to compare catestatin serum levels between the patients with a fatal outcome and those who survived. The secondary goal was to evaluate possible correlations between the serum catestatin levels and anthropometric, clinical and laboratory parameters of interest. Finally, having in mind the overlapping pathophysiological pathways of catestatin and both vitamin D and GDF-15, we aimed to explore the association between serum levels among the above-noted in the setting of severe COVID-19.This cross-sectional study was conducted at University Hospital of Split Respiratory Intensivist Center in the period from September 2021 to February 2022. This study was approved by the Ethics Committee at the University Hospital of Split , and it was performed according to the principles of the Declaration of Helsinki from 2013. Prior to the inclusion, all participants were informed about the purpose, course and aims of this study. Moreover, all of them signed a written informed consent form.2 < 50 mmHg or SpO2 < 90%) despite maximal noninvasive respiratory support. Included patients were treated by the up-to-date protocol in that period of time, and there were no differences in the approach of the treatment.The study included 152 patients treated in the intensive care unit (ICU) of the Respiratory Intensive Center. All enrolled patients were intubated and placed on mechanical ventilation on the first day of admission to the ICU. At the time of this study, the dominant variant of SARS-CoV-2 in Croatia was the delta variant (B.1.617.2). The standard references for endotracheal intubation included the following: airway protection, severe decompensate acidosis (pH < 7.2) and severe absolute hypoxemia  that were treated in the ICU because of positive PCR test on SARS-CoV-2; fatal outcome due to reasons other than COVID-19.All of the included participants underwent detailed physical examinations. Moreover, their medical history was thoroughly inspected regarding all of their personal and family anamnestic data. Clinical parameters used in the study analyses were measured on the day of the admission to the ICU. The following standard therapeutic methods and up-to-date protocols were used in patient treatment: corticosteroids, antiviral medications, anticoagulants, oxygen and other supportive therapies.Blood samples were collected on the day of the patients’ admission to the ICU. The samples were collected according to the standard laboratory practice. All of the hematological and biochemical parameters were analyzed on the same day while the samples for catestatin and GDF-15 determination were stored at −80 C° for later analyses.Serum catestatin levels were established by an enzyme-linked immunosorbent assay (ELISA), using a commercially available diagnostic kit . According to the manufacturer’s instructions, the kit measurement range was 0–100 ng/mL. Reported sensitivity for catestatin was 0.05 ng/mL with a linear range of 0.05–0.92 ng/mL. Cross-reactivity with endogenous human catestatin peptide for this assay kit was 100% with the intra-assay and inter-assay coefficients of variability being <10% and <15%, respectively. The samples were not diluted before the analyses.Serum levels of GDF-15 levels were determined using an electrochemiluminescence immunoassay on a Cobas e8000 analyzer . Reported sensitivity for GDF-15 was 400 pg/mL with a linear range of 400–20,000 pg/mL. The inter-assay coefficient of variability was 5%.All blood samples were managed according to the international standards, in the same laboratory, by the same experienced medical biochemist who was blinded to the subjects’ group in the study.t-test while the non-continuous variables were compared using the Mann–Whitney U test. Categorical variables were compared between groups using the chi-square test. Correlation was calculated using Pearson’s correlation for continuous variables and Spearman’s for the non-continuous variables. Additionally, independent predictors for COVID-19 survival were evaluated using the multivariate logistic regression, with OR (odds ratio), 95% CI  and p-value reported. The level of statistical significance was set at p < 0.05.Analyses of the collected data were conducted using the statistical software MedCalc . All continuous quantitative variables were presented as mean ± standard deviation while the non-continuous variables were presented as median (interquartile range). All qualitative variables were presented as whole numbers and percentages. The normality of distribution was estimated using Kolmogorov–Smirnov test. Comparison of continuous variables between groups was conducted using Student’s p = 0.031). There were no other significant differences between the non-fatal and the fatal group regarding the baseline characteristics (There were 105 (69.0%) patients with COVID-19 in the non-fatal group and 47 (31.0%) in the fatal group. The mean age of the study sample was 76.4 ± 8.6 years. There were significantly more smokers in the non-fatal group compared to the fatal group (35 (33.3%) vs. 7 (14.9%); eristics .9/L; p = 0.002), neutrophils , LDH  and hs-TnI (9.4 (6.0–14.1) vs. 18.6 (9.2–30.8) ng/L; p < 0.001) while the fatal group had a significantly lower level of lymphocytes (0.9 (0.6–1.3) vs. 0.7 (0.3–1.0) × 109/L; p = 0.002)  vs. 11.8 (8.1–14.8) × 10= 0.002) .p < 0.001)  .p < 0.001) while the GDF-15 levels were significantly higher in the non-fatal group   .p < 0.001) and significant negative correlation between serum catestatin levels and GDF-15 levels  (p = 0.036) and diastolic BP   . Moreove= 0.013) .p = 0.010) for a fatal COVID-19 outcome. On the other hand, serum GDF-15 levels  and leukocyte count  were the significant positive predictors for a fatal COVID-19 outcome. Other computed independent variables were age, gender and vitamin D levels  . It was As aforementioned, our results show a negative correlation between serum levels of catestatin and GDF-15. Previous studies have well established that GDF-15 has a major impact on the development and progress of numerous diseases due to its metabolic regulation . MoreoveAnother important result of this study is the positive correlation between serum catestatin and vitamin D levels. Vitamin D was thoroughly investigated regarding its implicated connection with a good outcome of the COVID-19 infection . It was There are several limitations to our study. We had a relatively small sample size, and it was conducted in a single center. Moreover, its cross-sectional design prevents making any causal conclusions. Even though catestatin is well researched and we are able to compare our results with other studies, we are still missing a healthy control group. In the present study, the viral load was not assessed, and such an aspect may be relevant for CST and GDF-15 serum concentrations. Lastly, we were not able to eliminate all of the confounding effects which could have possibly interfered with the results of the study.In conclusion, our study showed that patients with COVID-19 have a high level of catestatin. However, when comparing patients with the fatal outcome and those who survived, the latter have significantly higher serum catestatin levels. All of this implies that catestatin could be playing a major role in the complex pathophysiology of the COVID-19 infection. Moreover, the negative correlation between GDF-15 and our logistic regression model suggests that serum catestatin could also be a predictor of a poor COVID-19 outcome. However, all of the presented evidence should be addressed in a future larger-scale longitudinal multicentric study.